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Analysis of Volatile Constituents of Fermented Tea with Bacillus Subtilis

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Analysis of Volatile Constituents of Fermented Tea with Bacillus Subtilis Chiang Mai J. Sci. 2014; 41(2) 395 Chiang Mai J. Sci. 2014; 41(2) : 395-402 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper Analysis of Volatile Constituents of Fermented Tea with Bacillus subtilis by SPME-GC-MS Patcharee Pripdeevech*, Sakon Moonggoot, Siam Popluechai and Ekachai Chukeatirote School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand. *Author for correspondence; e-mail: [email protected] Received: 5 September 2012 Accepted: 12 December 2012 ABSTRACT The volatile components of Green Oolong tea No. 12 fermented with culture supernatants of five Bacillus subtilis strains were investigated. Initially, the culture supernatants of five different strains of B. subtilis were prepared and subsequently used as crude enzymes to ferment tea samples. After 2 h-fermentation, the volatile components were extracted using solid phase microextraction (SPME) technique and determined by gas chromatography-mass spectrometry (GC-MS). At least 54 components were identified in all samples. Linalool, hotrienol and γ-terpinene were found to be the major components in dry Green Oolong tea while B. subtilis-fermented teas provided 2-pentylfuran and limonene in higher amounts. The contents of most major volatiles increased remarkably in the fermented tea samples. Superior quantity of volatile components was related to the use of B. subtilis culture supernatants whereas 2-pentylfuran and limonene were responsible for the special odor of B. subtilis-fermented teas. Keywords: Camellia sinensis, Bacillus subtilis, SPME, GC-MS 1. INTRODUCTION Tea (Camellia sinensis) is a popular drink improve its aroma [12]. Other approaches worldwide and more than 3 million hectares include modification of the tea production has been planted with tea [1]. Tea is applied process (i.e., withering, rolling, and in pharmaceutical products [2-4]. Green tea fermentation) which result in aroma changes production does not involve fermentation by promoting and/or inhibiting the enzymes whereas Oolong and red tea are produced in the tea leaves [13,14]. Key odor compounds through semi-fermentation. Black tea is detected from these experiments showed that obtained though a complete fermentation monosaccharide or disaccharide flavorless process. The odors and flavors of tea result glycoside precursors were present in fresh tea from important components such as terpenes, leaves [15-21]. Free aroma constituents are caffeine, organic acids and polyphenols [5-11]. then released by hydrolysis of glycoside There have been many attempts to develop precursors by β-D-glycosidase enzymes new tea products especially those with distinct [13,14]. In addition, the addition of external aromas. One simple method is to include enzymes (i.e., pectinase and glucosidase) may edible essential oils into the tea product to improve tea aromas [12,22]. 396 Chiang Mai J. Sci. 2014; 41(2) Thua nao is a conventional fermented in this present study including B. subtilis TN51 soybean generally used as a flavor enhancer in isolated from thua nao, a Thai fermented dishes mainly in the northern part of Thailand. soybean [3], B. subtilis ASA and B. subtilis Cooked soybean is fermented with Bacillus BEST195 isolated from Japanese natto [3,31], subtilis and related bacilli [23]. It has been B. subtilis S1-13 isolated from terasi, an reported that Bacillus species are capable of Indonesia shrimp paste [32], and B. subtilis synthesis a wide range of enzymes that can TISTR008 obtained from Thailand Institute be used in industry [24]. A dramatic increase of Scientific and Technological Research of several volatile components was found in (TISTR). Each bacterial strain was routinely soybean fermentation when using this bacterial cultured on nutrient agar (NA) and, for stock strain as a starter culture [25-28]. Owens and culture, the 20% glycerol bacterial culture was co-workers [26] reported large amounts of prepared and stored at -20°C. For inoculum 3-hydroxy-2-butanone, 2, 5-dimethylpyrazine preparation, a single colony of each bacterial and trimethylpyrazine during fermentation of strain was subcultured to a test tube containing soy-daddawa. Ouoba et al. [29] also noted that 3 ml of nutrient broth (NB) and incubated the highest contents of pyrazines in African at 37°C for 24 h. One milliliter of the cell soumbala, fermented by pure-starter B. subtilis, suspension was then transferred to a flask were detected significantly. It is therefore containing 250 ml of NB and then incubated evident that enzymatic action from B. subtilis by shaking (170 rpm) at 37°C. After can increase the amounts of volatiles in approximately 24 h of incubation (the A600 different soybeans products. However, there values were ~ 1.0), the bacterial cells were is no report describing the application of harvested from the culture media by B. subtilis on tea. In order to develop and centrifugation (8,500 rpm at 4°C for 10 min). improve aroma quality in tea product, the aim The supernatant was then collected to a sterile of the present study is to investigate volatile media bottle and was used as crude enzymes odor components of B. subtilis-fermented teas for tea fermentation. Alternatively, the crude obtained from Chiang Rai province which is culture supernatants were kept at 4°C until one of best place for planting tea in Thailand required. [30]. 2.3 Fermentation of Tea 2. MATERIALS AND METHODS Tea sample was ground into very small 2.1 Tea Samples particles (almost a powder) using an electric Green Oolong tea No. 12 (Camellia sinensis grinder. For each fermentation process, var. sinensis) samples obtained from Boonrod one hundred grams of powdered tea was farm, Chiang Rai, Thailand was used in this inoculated with 100 ml of the various B. subtilis study. The sample was stored below 5°C supernatant. For mixture of B. subtilis TN51 prior to fermentation with culture supernatants and ASA, 100 ml of each strain was added of various Bacillus strains. Mixtures of C8 to into 100 g of various tea samples. All samples C19 n-alkanes were purchased from Merck were fermented with different B. subtilis strains (Darmstadt, Germany). for 2 h prior to extraction by SPME. The experiment was carried out in triplicate. 2.2 Bacterial Strains, Culture Conditions and Crude Extract Preparation 2.4 Analysis of Volatile Constituents Five strains of Bacillus subtilis were used - Solid-phase microextraction (SPME) Chiang Mai J. Sci. 2014; 41(2) 397 The SPME apparatus with a SPME fiber of their Kov t retention indices, relative assembly holding 1.0 cm fused-silica fibers to C8-C19 n-alkanes, and comparison of the was purchased from Supelco, Bellefonte, PA, mass spectra of individual components with USA. A 50/30 μm divinylbenzene-carboxen- the reference mass spectra in the Wiley 275 polydimethylsiloxane (DVB-CAR-PDMS) and NIST05 databases and 2007 [33] with fiber was selected to extract the volatile corresponding data of volatile flavor components from tea leaf fermented with components in tea. various Bacillus strains. The fiber was mounted in the manual SPME holder and 3. RESULTS AND DISCUSSION preconditioned for 2 h in a GC injection port The fingerprints of volatile components set at 250°C. For each extraction, the sample of dry Green Oolong tea No. 12 from bottle was equilibrated at room temperature Boonrod farm fermented with bacterium around 25°C for 2 h. By insertion through supernatants of various B. subtilis strains are the septum of the sample bottle, the fiber was present in Figure 1. Percentages of peak area then exposed to the sample headspace for of volatile compounds of Green Oolong tea 30 min prior to desorption of the volatiles No. 12 fermented with various B. subtilis are into the splitless injection port of the GC-MS summarized in Table 1. There Similar instrument for 5 min. characteristics of all B. subtilis-fermented teas were illustrated. Fifty-four volatiles were - Gas Chromatography-Mass Spectrometry identified among the Green Oolong tea (GC-MS) No. 12 samples. Increased amounts of most The volatile constituents of tea leaves volatile components occurred among different fermented with various Bacillus strains obtained B. subtilis-fermented teas as compared to the from the SPME extracts with DVB-CAR- dry tea sample. Linalool, hotrienol, γ-terpinene, PDMS fiber were analyzed using a Hewlett 2-pentylfuran, δ-3-carene and endo-fenchol Packard model HP6890 gas chromatograph were found to be the major components in (Agilent Technologies, Palo Alto, CA, USA). dry Green Oolong tea No. 12. Small amounts It was equipped with an HP-5MS (5% phenyl- of terpinolene, 1,8-cineole, cis-linalool oxide polymethylsiloxane) capillary column (30 m (furanoid), limonene and trans-isolimonene 0.25 mm i.d., film thickness 0.25 μm; were also detected. Tea fermented with culture Agilent Technologies, USA) interfaced to an supernatants of B. subtilis TN51 contained HP model 5973 mass-selective detector. The limonene, 2-pentylfuran, δ-3-carene, E-β- oven temperature was initially held at 40°C ocimene, hotrienol and linalool as the key and then increased by 2°C/min to 220°C. constituents, while monoterpene components The injector and detector temperatures were such as terpinolene, α-terpinene, trans- 250 and 280°C, respectively. Purified helium isolimonene, γ-terpinene, and allo-ocimene was used as the carrier gas at a flow rate of were minor components. The dominant 1 ml/min. EI mass spectra were collected at components of B. subtilis ASA-fermented tea 70 eV ionization voltages over the range of were 2-pentylfuran, limonene, linalool, m/z 29-300. The electron multiplier voltage hotrienol and δ-3-carene. They were was 1150 V. The ion source and quadrupole accompanied by the small amounts of E-β- temperatures were set at 230°C and 150°C, ocimene, terpinolene, trans-isolimonene, 1, respectively. Identification of volatile 8-cineole and caffeine.
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